Bottom Line:
The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive.We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence.Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

ABSTRACTTreatment of head and neck squamous cell carcinoma, HNSCC, often requires multimodal therapy, including radiation therapy. The efficacy of radiotherapy in controlling locoregional recurrence, the most frequent cause of death from HNSCC, is critically important for patient survival. One potential biomarker to determine radioresistance is TP53 whose alterations are predictive of poor radiation response. DNA-damaging reactive oxygen species (ROS) are a by-product of ionizing radiation that lead to the activation of p53, transcription of p21(cip1/waf1) and, in the case of wild-type TP53 HNSCC cells, cause senescence. The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive. For the first time, we show that persistent exposure to low levels of the ROS, hydrogen peroxide, leads to the long-term expression of p21 in HNSCC cells with a partially functional TP53, resulting in senescence. We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence. It is p21's ability to sustain elevated levels of ROS, in turn, that allows for a long-term oxidative stress, and ensures an active p53-p21-ROS signaling loop. Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

fig4: Long-term elevated ROS in cells with wtp53 is dependent on p21. (a) HN30 and HN31 cells were treated with 4-Gy ionizing radiation, while in suspension with DCFDA, then 15 min later, this initial burst of ROS was analyzed using flow cytometry. (b) Both HN30 and HN31 cells were exposed to 4 Gy, then were prepared for DCFDA staining and flow cytometry at indicated time points. (c) HN30 Lenti and HN30 shp21 cells were exposed to 4 Gy, then treated with the superoxide indicator, DHE, and collected for flow cytometry analysis at the late time point of 96 hrs. Error bars represent S.D. of each sample performed in triplicate. *P<0.05, by Student's t-test

Mentions:
Analysis of ROS levels revealed that HNSCC cells, regardless of p53 status, do have an initial burst of ROS 15 min after exposure to radiation, albeit at a much higher level in wtp53 cells (Figure 4a). Following the ROS levels for an extended time course showed that the initial ROS burst in wtp53 cells was not only sustained but became significantly elevated at the 96 h, when compared with the slight, nonsignificant increase seen in mutp53 cells (Figure 4b).

fig4: Long-term elevated ROS in cells with wtp53 is dependent on p21. (a) HN30 and HN31 cells were treated with 4-Gy ionizing radiation, while in suspension with DCFDA, then 15 min later, this initial burst of ROS was analyzed using flow cytometry. (b) Both HN30 and HN31 cells were exposed to 4 Gy, then were prepared for DCFDA staining and flow cytometry at indicated time points. (c) HN30 Lenti and HN30 shp21 cells were exposed to 4 Gy, then treated with the superoxide indicator, DHE, and collected for flow cytometry analysis at the late time point of 96 hrs. Error bars represent S.D. of each sample performed in triplicate. *P<0.05, by Student's t-test

Mentions:
Analysis of ROS levels revealed that HNSCC cells, regardless of p53 status, do have an initial burst of ROS 15 min after exposure to radiation, albeit at a much higher level in wtp53 cells (Figure 4a). Following the ROS levels for an extended time course showed that the initial ROS burst in wtp53 cells was not only sustained but became significantly elevated at the 96 h, when compared with the slight, nonsignificant increase seen in mutp53 cells (Figure 4b).

Bottom Line:
The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive.We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence.Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

ABSTRACTTreatment of head and neck squamous cell carcinoma, HNSCC, often requires multimodal therapy, including radiation therapy. The efficacy of radiotherapy in controlling locoregional recurrence, the most frequent cause of death from HNSCC, is critically important for patient survival. One potential biomarker to determine radioresistance is TP53 whose alterations are predictive of poor radiation response. DNA-damaging reactive oxygen species (ROS) are a by-product of ionizing radiation that lead to the activation of p53, transcription of p21(cip1/waf1) and, in the case of wild-type TP53 HNSCC cells, cause senescence. The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive. For the first time, we show that persistent exposure to low levels of the ROS, hydrogen peroxide, leads to the long-term expression of p21 in HNSCC cells with a partially functional TP53, resulting in senescence. We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence. It is p21's ability to sustain elevated levels of ROS, in turn, that allows for a long-term oxidative stress, and ensures an active p53-p21-ROS signaling loop. Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.